Background & objectives: Paceilomyces farinosus is an
entomogenous fungus with a powerful insecticidal activity against the
larvae of Lipidoptera, Coleoptera and Hymenoptera. However, the
hypoglycaemic activity of P. farinosus extract has not been studied.
This study was undertaken to investigate the hypoglycaemic and
anti-diabetic effects of P. farinosus (G30801) in rats with
streptozotocin (STZ)-induced diabetes given a high-fat and compared with
normal rats.

Name: Indian Journal of Medical Research Publisher: Indian Council of Medical Research Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Health Copyright: COPYRIGHT 2010 Indian Council of Medical
Research ISSN:0971-5916

It is estimated that 143 million people in the world live with
diabetes and this number will probably double by the year 2030 (1). Type
2 diabetes mellitus (T2DM) currently affects more than 200 million
individuals worldwide (2). The characteristic features of T2DM include
hyperglycaemia, near normal insulin levels, varying degrees of insulin
resistance, slightly raised levels of glucagons and almost no
ketoacidosis (3). Coronary artery disease (CAD) is markedly increased in
subjects with T2DM; dyslipidemia in T2DM is often characterized by
increased triglycerides, small, dense low-density lipoprotein (LDL), and
low concentrations of high-density lipoprotein (HDL) cholesterol (4).
Increased LDL and decreased HDL cholesterol have been shown to associate
with the development of CAD in T2DM (5). Management of these
complications represents a huge financial burden (6). Investigations
with oral anti-hyperglycaemic agents derived from plants used in
traditional medicine have shown these plants with good antidiabetic
activity (7,8). Conventional drugs used have rigid and multiple dosing
regimen, high-cost, and untoward effects (9). There are more than 1200
plant species broadly used in the treatment of DM and many of these
showed effective hypoglycaemic activity after laboratory testing (8).

P. farinosus an entomogenous fungus, is a powerful insecticidal
against the larvae of Lipidoptera, Coleoptera and Hymenoptera. Bioactive
metabolites of P. farinosus have been investigated for antioxidant and
anti-tumour properties10-13. Studies on hypoglycaemic activity using P.
farinosus polysaccharides are not yet reported. Thus the present study
was undertaken to study the hypoglycaemic effect of P. farinosus extract
from various fermented medium [soybean (S), black bean (B), and rice
(R)] in rats with streptozotocin-induced diabetes and fed with high fat
diet.

Material & Methods

The study was carried out at Department of Life Science, National
Dong-Hwa University, Hualien, Taiwan.

Microorganism and media: P. farinosus G30801 was kindly provided by
Prof. Lee Son-Tay (Department of Biotechnology, Southern Taiwan
University of Technology). The voucher specimens were deposited at the
culture collection laboratory of Department of Biotechnology, Southern
Taiwan University of Technology, Tainan County, Taiwan. P. farinosus
G30801 was initially grown on potato dextrose agar (PDA) medium in a
petri dish at 17[degrees]C, and then transferred to 250 ml flasks
containing 100 ml of seed culture potato dextrose agar broth (PDB)
medium and incubated on a rotary shaker (100 rpm) at 17[degrees]C for 7
days. Soybean (S), black bean (B), and rice (R) were evaluated for their
potential as the major solid substrate in solid-state fermentation
(SSF), respectively. Beans were soaked in 2-3 volume of water at room
temperature for 4 h. Rice was boiled in water at 100[degrees]C. SSF was
carried out by taking 300 g of the treated solid substrate in 1000 ml
wide-mouth plastic bottle, moistening with liquid media solution
containing 2 per cent peptone, 0.1 per cent K[H.sub.2]P[O.sub.4]
dissolved in distilled water. All plastic bottles were autoclaved at
121[degrees]C for 30 min and after cooling were inoculated with 10 ml of
seed culture and incubated at 17[degrees]C for 20 days. The fermented
product by SSF was freeze-dried (VirTis apparatus; Gardiner, NY, USA)
and then ground into flour as the tested sample. The fermented products
cultured from soybean, black bean and rice were named as S, B, and R
powder, respectively.

Quantification of crude water-soluble polysaccharides: The
fermented product of P. farinosus by solid-state fermentation was
extracted with water (100[degrees]C, 4 h). The supernatant was obtained
by centrifuging at 5000 xg to separate the solid from the crude extract.
The supernatant were extracted by Sevag method to remove the
dissociative protein (14). The supernatant was followed by three
consecutive precipitations with 4 volumes of 95 per cent ethyl alcohol
(v/v). The precipitated polysaccharides were dried under vacuum and the
crude polysaccharides were obtained. The polysaccharide content was
measured followed a phenolsulphuric acid method (15,16). The
polysaccharide content was 0.0335 g/g P. farinosus. The polysaccharide
content was taken as a token of index component for the quality control
of batch fermentation. The polysaccharide contents of fermented products
cultured from soybean (S), black bean (B), and rice (R) bore a
resemblance to the original one (data not shown).

Experimental animals: Wistar rats (n=70 body weight 250 [+ or -] 20
g at 8 wk old) were obtained from National Laboratory Animal Center,
Taipei, Taiwan. The rats were raised under a 12 h light/dark cycle and
had free access to food and water and maintained on a standard
laboratory diet (carbohydrates; 30%, proteins; 22%, lipids; 12%,
vitamins; 3%) ad libitum (17,18). The protocol of animal care was
recognized and approved from the Animal Ethics Committee of the
institution.

Induction of diabetes in rats: Rats (n=40) were fed a high fat diet
for 2 months and diabetes was induced by a single intraperitoneal
injection of freshly prepared streptozotocin (STZ, 30 or 50 mg/kg bw)
(Sigma, St. Louis, Mo, USA) in 0.1 M citrate buffer (pH 4.5) to
overnight fasted rats (modified from Zhang et al (19) via various dosage
for treating different types DM). After 2 wk of STZ administration,
animals with fasting blood glucose levels >200 mg/dl were considered
diabetic and included in this study.

Insulin-glucose tolerance test--To determine the response of the
diabetic rats to insulin action, they were injected with 0.5 g glucose
/kg bw ip, immediately followed by insulin (25.2 USP unit/mg, Sigma, MO,
USA) at a dose of 0.2 U/kg bw. At approximately 0, 30, 60, 90 and 120
min following insulin injection, blood was sampled by venipuncture from
the caudal vein and the percentage changes in blood glucose were
calculated for each group. The STZ-induced diabetic rats were followed
glucose tolerance test and insulin-glucose tolerance test (20) and
further defined as T1DM and T2DM based on amount of changed plasma
glucose.

Assessment anti-diabetic activity of P. farinosus extract from
various fermented medium in T1DM and T2DM rats--Two weeks after glucose
tolerance and glucose-insulin tolerance tests, normal (n=30), T1DM
(n=15) and T2DM (n=15) rats were fasted for 15 h and tested for blood
glucose. Rat was orally given tested sample (R powder, S powder or B
powder, 0.06 g/kg bw in water) and tested for blood glucose. After 30
min, rats were administrated (ip) 0.5 g glucose/kg bw. At approximately
0, 30, 60, 90 and 120 min following glucose injection, blood was sampled
by venipuncture from the caudal vein for determining glucose. The
percentage changes of glucose were calculated for each group.

Statistical analysis: The control and treatment groups were
compared by one-way ANOVA after performing the Duncan multiple range
tests.

Results

Rats fed with high fat diet showed increased body weight, CHOL,
plasma TG levels (Table I) as compared to the normal rats (P<0.05).
From the lipid profile it was evident that high fat fed rats showed
decreased LDL-C, and increased HDL-C levels as compared with the control
rats.

High-fat fed rat injected with STZ (30 and 50 mg/ kg bw) were found
to have (P<0.05) high TG levels in blood plasma. Contrary, high-fat
fed rat with STZ induction (50 mg/kg bw) showed the significantly
(P<0.05) higher percentage of HbA1c than that of the normal group
(Table II). As compared with normal rat, high-fat fed rats showed
significant difference (P<0.05) in the plasma total cholesterol.

To confirm the success of STZ-induced diabetes, rats were checked
for glucose tolerance and glucose-insulin tolerance tests, respectively,
at 2 wk following STZ administration. STZ-injected animals had blood
glucose exceeding 200 mg/dl, compared to a normal range of between 50
and 135 mg/dl following glucose injection at approximately 60 min in
relation to 0 min (Fig. 1). Further, insulin-glucose tolerance test
demonstrated that high-fat and STZ-induced rats had higher glucose level
following insulin administration (Fig. 2).

To further investigate the hypoglycaemic effects of P. farinosus
(G30801) fermented from various media (R, S and B) on diabetic rats, 30
min before glucose administration rats were treated with R, S or B
extracts. Single oral administration of R, S or B extracts of P.
farinosus (G30801) failed to alter the blood sugar in the normal rats
(P<0.05; Fig. 3). Compared to the normal rats, P. farinosus extract
from rice powder showed most significant hypoglycaemic effect at 120 min
in T2DM (P<0.01) and in T1DM (P<0.05) rats, respectively (Fig. 4
and 5).

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[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Discussion

The aim of the current work was to assess hypoglycaemic effect of P
farinosus in experimental diabetes model. Due to the high prevalence of
diabetes worldwide, extensive research is still being performed to
develop new antidiabetic agents and determine their mechanisms of
action, consequently, a number of diabetic animal models have been
developed and improved over the years (21).

Streptozotocin-induced diabetic rats are one of the animal models
of human insulin-dependent diabetes mellitus (22-24). As in human type 2
DM, diet has a great influence on the development of overt diabetes as
well as hypertension, hyperlipidaemia, and eventually nephropathy in
experimental models (25). In the present study, rats were fed with
high-fat diet and then induced with both low/high dose of STZ; high-fat
diet fed rats showed higher plasma TG, HDL-C, and lower LDL-C
concentration compared to normal rats. Similar phenomenon was previously
reported (26). The clinical symptoms of T2DM rats are closer to those of
diet and obesity related diabetes. STZ treatment induces weight loss
related to diabetes severity. The reduction of body weight might be due
to the low utilization of uptake blood sugar in cell.

Hyperlipidaemia has been reported to accompany hyperglycaemia
states (27), high levels of TC; importantly LDL cholesterol is one of
the major coronary risk factors (28) which is the major cause of
morbidity and deaths in diabetic subjects (29). A significance
difference (P<0.05) in the plasma total cholesterol was observed in
STZ induced diabetes rats as compared with the untreated group. The
present experimental data demonstrate that a high-fat diet rat can
successfully induce T2DM and T1DM via low/high dose STZ administration.